Biosynthesis of UDP-β-l-Arabinofuranoside for the Capsular Polysaccharides of Campylobacter jejuni

Campylobacter jejuni is the leading cause of food poisoning in North America and Europe. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) which enables adherence to the host epithelial cells and evasion of the host immune system. Many strains of C. jejuni can be differentiated from one another by changes in the sequence of the carbohydrates found within the CPS. The CPS structures of serotypes HS:15 and HS:41 of C. jejuni were chemically characterized and found to contain an l-arabinofuranoside moiety in the repeating CPS sequence. Sequence similarity and genome neighborhood networks were used to identify the putative gene cluster within the HS:15 serotype for the biosynthesis of the l-arabinofuranoside fragment. The first enzyme (HS:15.18) in the pathway was found to catalyze the NAD+-dependent oxidation of UDP-α-d-glucose to UDP-α-d-glucuronate, while the second enzyme (HS:15.19) catalyzes the NAD+-dependent decarboxylation of this product to form UDP-α-d-xylose. The UDP-α-d-xylose is then epimerized at C4 by the third enzyme (HS:15.17) to produce UDP-β-l-arabinopyranoside. In the last step, HS:15.16 catalyzes the FADH2-dependent conversion of UDP-β-l-arabinopyranoside into UDP-β-l-arabinofuranoside. The UDP-β-l-arabinopyranoside mutase catalyzed reaction was further interrogated by measurement of a positional isotope exchange reaction within [18O]-UDP-β-l-arabinopyranoside.

).The resonances labeled as H1 through H5 are the hydrogens for the glucuronate moiety of 4 while those resonances labeled as R2 through R5A/B are for the ribose moiety.The two hydrogens for the uridine moiety are found at 5.9 and 7.9 ppm and labelled as U5 and U6, respectively.The two hydrogens for the uridine moiety are found at 5.9 and 7.9 ppm are not shown in this spectrum.

Synthesis of Dibenzyl (2,3,5-tri-O-acetyl-β-L-arabinofuranoside) 1-phosphate (S5).
To a solution of 1.0 g (3.1 mmol) of 1,2,3,5-tetra-O-acetyl-D-arabinofuranose S3 in 5 mL of dry dichloromethane were added 3 mL of 33% hydrogenbromide in acetic acid and 0.2 mL of acetic anhydride; the solution was stirred at room temperature for 1 h.The solvent and the excess of reagent were evaporated in vacuo and the last traces removed by co-evaporation with toluene in vacuo.The residual bromide was dissolved in 5 mL of dry benzene and to the solution was added a benzene (3 mL) solution of 1.1 equivalent of triethylammonium dibenzyl phosphate (mixture of 1.1 equivalent of dibenzyl phosphate and 1.1 equivalent of triethylamine).After 1 h at room temperature, the crystalline triethylammonium bromide was filtered and washed with dry benzene.

Figure S1 :
Figure S1: Protein sequences of enzymes produced for this investigation.The polyhistidine purification tags are shown in yellow .

Figure S3 :
Figure S3: Chemoenzymatic synthesis of oxygen-18 labeled UDP-b-L-arabinose.Additional details are found in the Materials and Methods section of the paper.

Figure S4 :
Figure S4: Michaelis-Menten plots for the reaction catalyzed by UDP-a-D-glucose 6dehydrogenase (HS:15.18).(A) Variation of the concentration of UDP-glucose at a fixed concentration of 1.0 mM NAD + at pH 8.7.(B) Variation of the concentration of NAD+ at a fixed concentration of UDP-glucose of 1.0 mM at pH 8.7.(C) Variation of the concentration of UDP-glucose at a fixed concentration of NAD+ of 1.0 mM at pH 8.0.All reactions contained 1.0 mM DTT.

Figure S5: 1 H
Figure S5: 1 H NMR spectrum of UDP-a-D-glucuronate (4).The resonances labeled as H1 through H5 are the hydrogens for the glucuronate moiety of 4 while those resonances labeled as R2 through R5A/B are for the ribose moiety.The two hydrogens for the uridine moiety are found at 5.9 and 7.9 ppm and labelled as U5 and U6, respectively.

Figure S6 :
Figure S6: Proposed mechanism for decarboxylation of UDP-a-D-glucuronate (4) to UDPa-D-xylose (5).In the proposed mechanism the NAD + is used to oxidize C4 to form a b-keto acid intermediate.After decarboxylation the keto-group is reduced by the newly formed NADH. 1

Figure S7: 1 H
Figure S7: 1 H NMR spectra of UDP-a-D-xylose (5).(A) UDP-a-D-xylose formed in H2O by the catalytic activity of UDP-a-D-glucuronate 6-decarboxylase using UDP-a-D-glucuronate (4) as the substrate.(B) UDP-a-D-xylose formed in D2O using UDP-a-D-glucuronate (4) as the substrate.The resonances labeled as H1 through H5 are the hydrogens for the xylose moiety of 5 while those resonances labeled as R2 through R5A/B are for the ribose moiety.The two hydrogens for the uridine moiety are found at 5.9 and 7.9 ppm are not shown in this spectrum.

Figure S8 :
Figure S8: Michaelis-Menten curves for variation of substrate concentration for the reaction catalyzed by UDP-a-D-glucuronate 6-decarboxylase (HS:15.19).(A) Variation of UDP-D-glucuronate at a fixed concentration of 0.3 mM NAD + at pH 6.5.(B) Variation of the concentration of NAD + at a fixed concentration of 2.5 mM UDP-D-glucuronate at pH 6.5.

Figure S9: 1 H
Figure S9: 1 H NMR spectra of an equilibrium mixture of UDP-a-D-xylose (5) and UDP-b-Larabinose (6) formed in H2O and D2O.(A) UDP-b-L-arabinose formed in H2O by the catalytic activity of UDP-a-D-xylose 4-epimerase using UDP-a-D-xylose (5) as the initial substrate.(B) Formation of the product in D2O.The resonances labeled as HA1 through HA5 are the hydrogens for the L-arabinose moiety of 6 while those resonances labeled as R2 through R5A/B are for the ribose moiety.The resonance labeled as HX1 through HX5 are the hydrogens for the D-xylose moiety.The two hydrogens for the uridine moiety are found at 5.9 and 7.9 ppm and are not shown in this spectrum.

Figure S13 :
Figure S13: 13 C spectrum of the a/b mixture of S2 in MeOH-d4.

Figure S14: 1
Figure S14: 1 H spectrum of an a/b mixture of compound S3 in CDCl3.

Figure S15 :
Figure S15: 13 C spectrum of an a/b mixture of compound S3 in CDCl3

Figure S19: 1 H
Figure S19: 1 H spectrum of 7 in D2O.The resonance at 1.20 ppm and 3.12 ppm are from triethyl amine.